Hepatitis

ABSTRACT

A screening method of identifying a compound for treating hepatitis. Also disclosed is a method for evaluating responsiveness of a subject having hepatitis to a drug.

RELATED APPLICATIONS

This application is a divisional application and claims priority to U.S.application Ser. No. 10/688,302, filed Oct. 15, 2003, the contents ofwhich are incorporated herein by reference.

BACKGROUND

Hepatitis can be caused by many infection agents, including hepatitis A,B, C, D, and E viruses and GB virus.

Viral hepatitis is the single most important cause of liver disease.Take hepatitis C for example, it is estimated to affect 170 millionpeople worldwide. Patients with liver damage resulting from hepatitis Cmay develop chronic liver diseases, such as cirrhosis and hepatocellularcarcinoma. Hepatitis C can be treated with interferon α. However, onlyabout 50% hepatitis C patients are responsive to the treatment. Otherdrawbacks to interferon α therapy include significant side effects, highcosts, and poor responsiveness to hepatitis C virus genotype 1, the mostcommon genotype in the United States. New therapies have beingvigorously sought. Although several drug candidates are now beingevaluated, the progress is rather slow due to a lack of appropriateanimal models and uncertainty of responsiveness in humans.

Thus, there is a need for a reliable method for identifying drugs fortreating hepatitis C and other viral hepatitis.

SUMMARY

This invention is based, at least in part, on an unexpected discoverythat hepatitis virus replicates in vitro in human liver slices. Liverslices infected with hepatitis virus can thus be used to screencompounds for treating hepatitis.

One aspect of the invention features a screening method of identifying acompound for treating hepatitis. The method includes (1) obtaining afirst liver slice from a subject having hepatitis; (2) incubating thefirst liver slice in a medium containing a compound; and (3) determiningthe replication level of a hepatitis virus, e.g., a hepatitis C virus,hepatitis B virus, hepatitis D virus, or GB virus, in the first liverslice. The compound is determined to be effective in treating hepatitisif the replication level of the hepatitis virus is lower than thatdetermined in the same manner from a second liver slice except that thesecond liver slice is incubated in a medium free of the compound. Thereplication level of the hepatitis virus can be based on the replicationrate, genome level, or protein level of the hepatitis virus. In apreferred embodiment, the method is used to identify a compound fortreating hepatitis C. In this embodiment, the protein level of HCV canbe that of core protein, E1 protein, E2 protein, p7 protein, NS3protein, NS4A protein, NS4B protein, NS5A protein, NS5B protein, or Fprotein.

It is known that hepatitis patients respond differently to differentanti-hepatitis drugs. The invention also features a method of evaluatingresponsiveness of a subject having hepatitis to a known drug. Thismethod is identical to the method described above, except that a knowndrug, instead of a test compound, is used. The subject is determined tobe responsive to the drug if the replication level of the hepatitisvirus in the first slice is lower than that in the second slice. Thismethod can be employed before, during, or after a therapy.

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Other advantages, features, andobjects of the invention will be apparent from the detailed descriptionand the claims.

DETAILED DESCRIPTION

This invention relates to the use of liver slices in identifyingcompounds in treating hepatitis.

For example, within the scope of this invention is a method forscreening a compound that can be used in treating hepatitis. To practicethe method, one incubates a compound in a medium with a liver sliceprepared from a subject having hepatitis for a period of time, e.g., 24to 96 hours, and then determines the replication level of the virus,such as genome level, protein level, or the replication rate of thevirus, in the liver slice. One also determines a control replicationlevel of the virus in a second liver slice in the same manner exceptthat the second liver slice is incubated in a medium free of thecompound. If the replication level in the first slice is lower than thatin the second slice, the compound is a candidate for treating hepatitis.

A liver slice can be prepared using techniques well known in the art. Itcan be prepared in different dimensions and maintained in variousculture systems. See, e.g., Groneberg et al., Toxicol. Pathol. 30 (2002)394-399 and Ekins Drug Metab. Rev. 28 (1996) 591-623. A plurality ofliver slices can be obtained from a subject and stored in, e.g., liquidnitrogen, for later use (Isachenko et al., Eur. J. Obstet. Gynecol.Reprod. Biol. Jun. 10, 2003;108(2):186-93). These slices can also beused in parallel to screen different compounds, thereby achievinghigh-throughput screening.

The replication level of a virus can be determined using techniquesdescribed in the example below or using those known in the art. Forexample, the genome level can be determined using RT-PCR. To determinethe level of a viral protein, one can use techniques including ELISA,immunoprecipitation, immunofluorescence, EIA, RIA, and Western blottinganalysis. To determine the replication rate of a virus, one can use themethod described in, e.g., Billaus et al., Virology 26 (2000) 180-188.

Compounds to be screened can be obtained using any of the numerousapproaches in combinatorial library methods known in the art. Suchlibraries include: peptide libraries, peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone that is resistant to enzymatic degradation),spatially addressable parallel solid phase or solution phase libraries,synthetic libraries obtained by deconvolution or affinity chromatographyselection, the “one-bead one-compound” libraries, and antibodylibraries. See, e.g., Zuckermann et al. (1994) J. Med. Chem. 37,2678-85; Lam (1997) Anticancer Drug Des. 12, 145; Lam et al. (1991)Nature 354, 82; Houghten et al. (1991) Nature 354, 84; and Songyang etal. (1993) Cell 72, 767. Examples of methods for the synthesis ofmolecular libraries can be found in the art, for example, in: DeWitt etal. (1993) Proc. Natl. Acad. Sci. USA 90, 6909; Erb et al. (1994) Proc.Natl. Acad. Sci. USA 91, 11422; Zuckermann et al. (1994) J. Med. Chem.37, 2678; Cho et al. (1993) Science 261, 1303; Carrell et al. (1994)Angew. Chem. Int. Ed. Engl. 33, 2059; Carell et al. (1994) Angew. Chem.Int. Ed. Engl. 33, 2061; and Gallop et al. (1994) J. Med. Chem. 37,1233.Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13, 412-421), or on beads (Lam (1991) Nature 354,82-84), chips (Fodor (1993) Nature 364, 555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. No. 5,223,409), plasmids (Cull et al.(1992) Proc. Natl. Acad. Sci. USA 89, 1865-1869), or phages (Scott andSmith (1990) Science 249, 386-390; Devlin (1990) Science 249, 404-406;Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382; Felici(1991) J. Mol. Biol. 222, 301-310; and U.S. Pat. No. 5,223,409).

Compounds to be screened can be known drugs that are used to treat otherdiseases. Those that are found to inhibit hepatitis virus replication inliver slices can be used in treating hepatitis. Screening known drugs isadvantageous, since the toxicity, pharmacokinetics, and side effectsdata of the drugs are available and all ethical issues have already beensolved. The only remaining issue is whether the drugs are effective intreating hepatitis. Take sodium stibogluconate for example. A compoundof pentavalent antimony, it has been used to treat kala-azar as thefirst-line medicine for more than 50 years (Herwaldt et al., Am. J.Trop. Med. Hyg. 46 (1992) 296-306). It is administrated by intravenousor intramuscular injection at a daily dose of 20 mg/kg for 28 days. Asshown in the example below, this drug suppresses hepatitis C virus (HCV)replication in liver slices obtained from hepatitis C patients. As theeffective blood concentration of this drug for inhibiting HCVreplication, e.g., 100 μg/ml, is much lower than what is needed fortreating kala-azar (400 μg/ml), the drug is expected to be safe intreating hepatitis C.

The invention also features a method of evaluating an individual'sresponsivenss to an anti-hepatitis drug. Hepatitis patients showdifferent responsiveness to a therapy. For example, interferon-basedtherapies can only achieve a sustained virological response in about 50%of HCV patients (McHutchison et al., Clin. Liver Dis. 7 (2003) 149-161).As the above-described screening method is based on liver slicesprepared from individual subjects, it can be used to examine theresponsiveness of a patient to a drug and thereby determine whether thedrug is suitable for treating that patient.

To evaluate a subject's responsiveness to different drugs, one preparesa number of liver slices from the subject and incubates them with thedrugs, respectively. One then obtains the replication level of the virusin each of the liver slices and compares it to a control level in themanner described above. The subject is determined to be responsive tothe drug if the replication level in a slice incubated with a drug islower than the control level. This method can be used to monitor ahepatitis treatment in a subject. For this purpose, liver slices areprepared from a subject before, during, and after undergoing treatment.The slices are then subjected to the treatment in vitro, and thereplication level of the virus in each slice is obtained in the mannerdescribed above. The subject is determined to have developed resistanceto treatment, if the viral replication level in liver slices preparedduring or after the treatment is higher than that in slices preparedbefore the treatment. Such information is useful in prognostication forhepatitis. It also assists clinicians in designing other therapies.

The specific example below is to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications cited herein arehereby incorporated by reference in their entirety.

Patients

Under informed consent, biopsy samples were obtained from 5 groups ofpatients (i.e., Groups I-V). Each patient was tested for presence ofanti-HCV antibody (“Anti- HCV”) and hepatitis B surface antigen(“HBsAg”), the alanine aminotransferase activity (“ALT”), the knodellhistology activity index (“HAI”), the viral genotype (“Genotype”), andthe serum HCV-RNA concentration (“Concentration”) using standardtechniques. The basic information for each patient was listed in Tablebelow. TABLE Concen- tration ALT (10⁶ Age Anti- (Unit/ HAI Geno- copies/Patients Sex (year) HCV HBsAg Liter) score type ml) Group I. Biopsytissues treated with IFN IFN-1 M 37 P N 698 7 1b <0.01 IFN-2 F 49 P N101 7 2b 20.35 IFN-3 M 37 P P 111 5 2a <0.01 IFN-4 M 54 P N 47 5 1b 1.70IFN-5 F 48 P P 370 8 1b 31.01 IFN-6 F 40 N P 282 5 — — Group II. Biopsytissues treated with inactivated IFN iFN-1 M 51 P N 147 3 1b 39.01 iFN-2M 47 P N 368 8 1b 45.51 iFN-3 F 52 P N 326 11 1b 2.82 iFN-4 M 53 P P 1124 2a <0.01 iFN-5 M 47 N P 633 8 — — iFN-6 F 60 P P 121 9 2a 2.90 GroupIII. Biopsy tissues treated with 10 μg/ml of Stibogluconate S10-1 F 52 PN 253 6 1b 0.49 S10-2 M 39 P N 125 6 2a 0.07 S10-3 F 42 WP N 23 3 1b<0.01 S10-4 M 40 P N 63 1 2a <0.01 S10-5 M 42 N P 58 9 — — S10-6 M 53 NN 949 4 1b 0.15 Group IV. Biopsy tissues treated with 100 μg/ml ofStibogluconate S00-1 F 52 P N 145 8 2b 41.01 S00-2 M 56 P N 89 3 1b<0.01 S00-3 M 43 P N 39 7 1b <0.01 S00-4 F 55 P N 90 10 2a 0.85 S00-5 F44 P N 88 7 1b 45.30 S00-6 M 25 P N 75 3 1b 98.33 Group V. Biopsytissues used for viability tests V-1 F 75 P N 251 12 1b 0.14 V-2 F 76 NN 253 4 2a 3.50 V-3 M 48 P N 89 8 1b 26.28 V-4 M 47 P P 112 10 2a <0.01V-5 F 58 P N 147 6 1b <0.01 V-6 F 38 P N 134 5 1b <0.01

All patients were positive for anti-HCV antibody except IFN-6, iIF-5,S10-5, S10-6, and V-2. Among these 5 patients, S10-6 and V-2 werepositive for serum HCV-RNA and diagnosed as having acute hepatitis C.The other three patients were tested positive for hepatitis B surfaceantigen and diagnosed as having chronic hepatitis B, and were includedas negative controls. No statistically significant difference was foundamong the five groups in sex, age, ALT activity, HAI score, viralgenotype, or serum HCV-RNA concentration.

Liver Slices

Liver slices were prepared from liver tissues of the patients. To obtainthe livers tissues, biopsy was performed using Bard Biopty-Cut biopsyneedles (C. R. Bard, Inc. Covington, Ga.) with a diameter of 1.2 mm (18gauge). Two equal-sized slices, 1 mm in thickness, were separated fromeach tissue. The remaining portion (main portion) of each tissue wassubjected to pathological examination. The liver slices were incubatedin a 24-well culture plate containing 2 ml of minimal essentialmedium-20% fetal bovine serum and fed with fresh medium daily.

Drug Evaluation

1. Sodium stibogluconate suppressed HCV replication in human liver slice

Sodium stibogluconate had been found capable of suppressing thereplication of an HCV replicon in a HCV subgenomic RNA replicon system(Ava5; Apath; St. Louis, Mo.). To evaluate whether it suppresses theauthentic HCV replication in liver slices, the liver slices frompatients S10-1 to S10-6 and SOO-1 to SOO-6 were incubated with 0, 10,and 100 μg/ml of sodium stibogluconate (Wuhan Shengmao Corp., Hubei,China), respectively, 24 hrs after the slices had been cultured invitro. The slices from patient S10-5 served as a negative control. Threedays later (i.e., 96 hr after the slices were cultured), the level ofHCV RNA in each slice was measured.

To detect the HCV-RNA level, one step RT-PCR (not nested) was performedin the manner described in Yeh et al., J. Gen. Virol. 78 (1997)2761-2770. As a control, β-actin mRNA was measured simultaneously. 20PCR cycles were performed, in which the amounts of the PCR products hadnot yet reached to a plateau. The primers used were described in Yeh etal., Oncogene 19 (2000) 5213-5220. The HCV-RNA was quantified accordingto a method comparable to the branched DNA method described in Yeh etal, J. Virol. Methods 65 (1997) 219-226. HCV genotypes were determinedusing Inno-Lipa HCV II kit (Innogenetics, Zwijndrecht, Belgium).

It was found that, at 10 μg/ml, sodium stibogluconate partiallysuppressed HCV replication in the slices from patients S10-1, S10-2,S10-4, and S10-6, but not in those from patient S10-3. At 100 μg/ml, italmost completely suppressed HCV replication in the slices from patientsS00-1, S00-2, S00-3, and S00-5, but not in those from patients S00-4 andS00-6.

2. Sodium stibogluconate suppressed HCV replication in 293EBNA-Sip-Lcell line

To verify the ability of sodium stibogluconate to inhibit HCVreplication, a 293EBNA-Sip-L cell line was used. This cell line,permissive for HCV infection and replication, had been established byexpressing a cellular factor, Sip-L, in 293EBNA cells (Yeh et al., J.Virol. 75 (2001) 11017-11024). This cell line was maintained in aDulbecco's modified Eagle's medium containing 10% fetal bovine serum,250 μg/ml G418, and 150 μg /ml hygromycin B. The cells were infectedwith HCV. Briefly, cells in a 60-mm-diameter petri dish were incubatedwith a medium containing 5 μl of HCV-positive serum (10⁷ copies ofHCV-RNA/ml) for 12 hr. The cells were then incubated in a fresh mediumwithout the serum.

At the second day after the HCV infection, 293EBNA-Sip-L cells wereincubated with media containing 0, 1, 10, and 100 μg/ml of sodiumstibogluconate, respectively. At the seventh day, the cells wereharvested to determine the intracellular level of HCV-RNA. The cellswere trypsinized and washed two times with a fresh medium bycentrifugation. The supernatant of the second wash (as a contaminationcontrol) and the washed cells were collected in pairs for RNA extractionand nested RT-PCR. A digoxigenin-labeled probe was used for thesubsequent Southern blot analysis. The sequences of the primers, themethod for generating the probe, and the procedure for RT-PCR weredescribed in Yeh et al., J. Virol. 75 (2001) 11017-11024. An amplifiedHCV fragment was cloned into pCR2.1 -TOPO (Invitrogen, Carlsbad, Calif.)to generate a plasmid, pTOPO-HCV5n, which served as a positive controlin Southern blot analysis. As internal controls, the levels of 18s and28s RNAs were measured.

Intracellular HCV-RNA in the cells not treated with sodiumstibogluconate was found to be 2.31×10 per 10⁵ cells. In contrast, nonewas detected in cells treated with 1, 10, or 100 μg/ml of sodiumstibogluconate using RT-PCR, which has a sensitivity of <10² copy per10⁵ cells.

Sodium stibogluconate has been used in treating kala-azar at a dailydose of 20 mg/kg. The estimated blood concentration immediately afterinjection is about 400 μg/ml, much higher than an effectiveconcentration found in this example (100 μg/ml). Thus, if the dosageused to treat kala-azar is given to hepatitis C patients, the drugconcentrations can be maintained above effective level for over twohalf-lives. Furthermore, at a concentration as low as 10 μg/ml, sodiumstibogluconate partially suppresses HCV replication. These indicate thatsodium stibogluconate can be used to treat hepatitis C.

3. Responsiveness of patients to interferon-alpha 2b

The responsiveness of patients to Interferon-alpha, a drug currentlybeing used to treat hepatitis C, was evaluated using liver slices. Liverslices prepared from patients INF-1 to 6 were incubated with 5000 U/mlof interferon-alpha2b (Schering-Plough Corp., Kenilworth, N.J. Slicesfrom patient IFN-6, who was infected by hepatitis B virus, were used asnegative controls.

It was found that the HCV-RNA levels in the liver slices from patientsIFN-1, 3, and 4 were suppressed to undetectable levels. Partialsuppression was observed in the slices from patient IFN-5, and nosuppression was seen in those from patient IFN-2. Judging from cellviability results and the β-actin levels in all slices, the differencesin the responses to interferon-alpha were not due to cell damages ortissue sizes.

To prove that the interferon-alpha used was active, it was heated to 90°C. for 10 min before being incubated with the slices from patients ilF-1to 6 in the same manner described above. The slices form patient iIF-5served as negative controls. No suppression was found in these slices,indicating that the interferon-alpha was active.

4. Sodium stibogluconate and interferon-alpha 2b each protectedhepatocytes

To determine whether sodium stibogluconate or interferon-alpha 2bprotects hepatocytes in human liver slices, the viability of hepatocytesin human liver slices incubated with the drugs was examined. As HCVreplication causes the death of hepatocytes, intracellular aspartateaminotransferase (AST) leaks out. The intracellular level of ASTtherefore positively correlates with the cell viability.

Liver slices prepared from the patients in Group V were treated withsodium stibogluconate or interferon-alpha 2b in the manner describedabove. The AST activity in the medium was determined using a standardtechnique 48, 72, and 96 hrs after the slices had been cultured invitro. The AST activities in a fresh medium and in the liver slices 96hrs after incubation were also measured. For this purpose, the sliceswere homogenized using a mini-homogenizer (Pellet Pestles, KimbleDeltaware, Vineland, N.J). The percentages of AST leaked out to themedium and remained in the liver slices were calculated accordingly. TheAST-leakage in the first 24 hr was measured as the liver cell damage atthis stage was mostly caused by separation of the liver slices from thebiopsy tissues.

It was found that, 96 hrs after the culture, the intracellular ASTlevels in cells incubated with sodium stibogluconate andinterferon-alpha 2b decreased progressively to 72% and 83%,respectively. In cells not incubated with any drugs, the intracellularAST levels decreased to 57%. No significant difference was found betweenany two samples obtained at the same time-point. The results indicatedthat sodium stibogluconate and interferon-alpha 2b protectedhepatocytes.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of thefollowing claims.

1. A screening method of identifying a compound for treating hepatitis,the method comprising: obtaining a first liver slice from a subjecthaving hepatitis; incubating the first liver slice in a mediumcontaining a compound; and determining the replication level of ahepatitis virus in the first liver slice, wherein the compound iseffective in treating hepatitis if the replication level of thehepatitis virus is lower than that determined in the same manner from asecond liver slice obtained and incubated in the same manner as thefirst liver slice except that the second liver slice is incubated in amedium free of the compound.
 2. The method of claim 1, wherein thereplication level of the hepatitis virus is based on the replicationrate of the hepatitis virus.
 3. The method of claim 1, wherein thereplication level of the hepatitis virus is based on the genome level ofthe hepatitis virus.
 4. The method of claim 1, wherein the replicationlevel of the hepatitis virus is based on the protein level of thehepatitis virus.
 5. The method of claim 1, wherein the hepatitis virusis a hepatitis C virus, hepatitis B virus, hepatitis D virus, or GBvirus.
 6. The method of claim 5, wherein the replication level of thehepatitis virus is based on the replication rate of the hepatitis virus.7. The method of claim 5, wherein the replication level of the hepatitisvirus is based on the genome level of the hepatitis virus.
 8. The methodof claim 5, wherein the replication level of the hepatitis virus isbased on the protein level of the hepatitis virus.
 9. The method ofclaim 5, wherein the hepatitis virus is a hepatitis C virus,
 10. Themethod of claim 9, wherein the replication level of the hepatitis virusis based on the replication rate of the hepatitis virus.
 11. The methodof claim 9, wherein the replication level of the hepatitis virus isbased on the genome level of the hepatitis virus.
 12. The method ofclaim 9, wherein the replication level of the hepatitis virus is basedon the protein level of the hepatitis virus.
 13. The method of claim 12,wherein the protein level is the level of the core protein, E1 protein,E2 protein, p7 protein, NS3 protein, NS4A protein, NS4B protein, NS5Aprotein, NS5B protein, or F protein of the hepatitis C virus. 14-26.(canceled).